Project Summary Osteoarthritis (OA) is a debilitating degenerative disease that afflicts an estimated 27 million Americans age 25 and older. This disease leads to the progressive degradation of the articular layers of diarthrodial joints, significantly compromising the main function of cartilage as a load bearing material, leading to pain and limiting activities of daily living. To this day, there are very limited treatment options for slowing down the progression of OA in its early stages. Most therapies, such as highly invasive partial and total joint replacement surgeries are performed at the late stage of the disease. Introducing treatment options to earlier stages of OA presents the potential to retard or slow down disease progression and thus significantly improve patient outcomes. The primary function of articular cartilage is to transmit loads across the joint surfaces while simultaneously minimizing friction and wear. This application describes the development of an ultrafast laser-based treatment tool which has the ability to induce crosslinks into the cartilage collagen network without the addition of a chemical agent, while simultaneously avoiding damaging effects of optical breakdown and ablation. Preliminary data show that laser-induced crosslinks increase compressive stiffness and wear resistance, without compromising cell viability, which may be expected to slow down progression of OA. The overall aim of this application is to develop a range of effective and safe laser operating parameters that enhance cartilage mechanical properties and wear resistance, enabling us to produce a clinically relevant protocol. We also aim to assess the influence of laser-induced short-lived bursts of reactive oxygen species onto the long-term response of cartilage during in vitro and in vivo culture. To translate this technology to future animal and human studies, we will develop and test a laser-based clinical tool for arthroscopic treatment of cartilage in situ. In specific aim 1, we will optimize a framework for structural, morphological and functional modification of the cartilage extracellular matrix subject to femtosecond laser irradiation, using devitalized bovine and human OA cartilage explants. In specific aim 2, we will narrow this range of operating parameters by testing short-term and long-term viability of laser-treated live bovine and human OA (male & female) cartilage explants against untreated controls, using in vitro culture up to 4 weeks. In specific aim 3, we will verify that laser-treated live human OA cartilage explants exhibit comparable viability and health as untreated controls when implanted for up to 8 weeks in the back of nude mice. We will also fabricate a fiber optic-based laser system and validate its effectiveness in simulated in situ arthroscopic applications in OA knee joints. Upon completion of these studies, we will have established effective and safe operating parameters for this novel laser treatment modality, and created a practical tool to test this methodology in situ, first in large animals, then in humans.